Numerous clinical and research situations necessitate highly sensitive analytical methods for the analysis of amino acids, peptides, amino sugars and other primary amine bearing compounds. During the last twenty years a number of separation procedures have been developed allowing resolution of the components of a complex mixture of primary amines. Furthermore, methods of utilizing laser-induced fluorescence detection have been developed and refined, permitting detection of exceedingly small amounts of a fluorescent product. A continuing problem in the area of primary amine detection has been the design of reagents that not only react well with a variety of primary amines, but also yield a product which is readily detected in low amounts and is easily separable from a complex mixture of reactants and products.
A number of derivatizing reagents have been developed which add a chromophore, detectable at a specific wavelength of light, or a fluorophore which may be excited by light at a specific wavelength to emit light at a different wavelength. Some of these reagents include ninhydrin, fluorescamine, 7-chloro-4-nitro-benzene-2-oxa-1,3-diazole (NBD-chloride) and ortho-phthalaldehyde (OPA) (Rubenstein et al., 1979, Anal. Biochem. 95:117-121; Stein et al., 1974, Arch. Biochem. Biophys. 163:400-403; De Bernado et al., 1974, Arch. Biochem. Biophys. 163:390-399; Ghosh et al., 1968, Biochem. J. 108:155-160; Roth, 1971, Anal. Chem. 43:880-882; Lindroth et al., 1979, Anal. Chem 51:1667-1674; Hodgin, 1979, J. Liq. Chromatogr. 2:1047-1059; and Deyl et al., 1986, J. Chromatogr. 379:177-250). OPA has been a popular reagent due to its ability to rapidly form intensely fluorescent isoindole products from non-fluorescent starting materials. However, the sensitivity of OPA analysis has been limited to the picomole range, and OPA derivatives are unstable due to their sensitivity to light, air oxidation and attack by acids (White et al., 1969, Adv. Heterocycl. Chem. 10:113-147). A newly developed reagent, naphthalene-2,3-dicarboxaldehyde (NDA) is structurally similar to OPA in that it has an ortho-dialdehyde moiety and it forms a fluorogenic isoindole product with a primary amine (De Montigny, 1987, Anal. Chem. 59:1096-1101). NDA forms a more stable isoindole product than OPA. The improved stability is partly due to the use of cyanide instead of thiol as a nucleophilic reagent, resulting in a nitrile group instead of a thiol group on the isoindole ring.
The above reagents usually allow detection in the picogram range when used with conventional separation systems such as liquid chromatography or high pressure liquid chromatography (HPLC) and non-laser fluorescence detection schemes. Sensitivity on this scale is inadequate for many current applications including, for example, the analysis of protein hydrolysates resulting from state-of-the-art protein microisolations. Improved separation techniques, such as capillary electrophoresis or capillary zone electrophoresis, especially when coupled with laser fluorescence detection, can increase the sensitivity of detection. However, in order to be useful for laser fluorescence detection, reagents must be developed which yield a product with an amine that is excitable by readily available lasers, for example the helium/cadium laser or the argon ion laser.
To form isoindole products that are easily excitable at the output wavelengths of readily available lasers, a number of reagents have been synthesized, including unsubstituted 3-benzoyl-2-quinoline-carboxaldehyde (Beale, et al., 1989, Talanta 36:321-325) and 3-(2-furoyl) quinoline-2-carboxaldehyde (Beale, et al., 1990, J. Chromatogr. 499:579-587). While such reagents are fairly versatile when used in conjunction with liquid chromatography separation techniques, their utility with preferred separation techniques such as capillary electrophoresis is limited. This is apparently due to the relatively hydrophobic nature of the resulting isoindole products. Another significant problem with these reagents is that they also fail to react with most peptides consisting of more than three amino acids and with many amino sugars.
Hence, there is an outstanding need for reagents which 1) have the capability of reacting with large peptides, proteins and amino sugars, 2) yield products that are separable by modern micro-separation procedures, such as capillary electrophoresis, and 3) are detectable by commonly used laser detection systems. The XArQCA reagents of the present invention meet these three criteria.